Antisense oligonucleotides specifically target a messenger RNA. They have found a wide range of in vitro functions including roles in diagnostics and research. They are also a potentially important class of novel therapeutics, with several in clinical trials.
In order to find its target in vitro, an oligonucleotide must enter the cell and localize to its target in the cytoplasm and/or nucleus. Unmodified oligonucleotides have demonstrated poor ability to cross cell membranes. Several approaches to enhance the uptake of oligonucleotides have been tried, including liposomal encapsulation, conjugation with other ligands, such as cholesterol, peptides and poly-lysines, and cationic lipids. Cationic lipids have been shown to increase the cellular uptake of oligonucleotides, but the mechanism by which this occurs is not well understood (Bennett, C. F., et al., Mol. Pharmacol. 1992 41, 1023-1033).
Oligonucleotide uptake is thought to proceed initially by interaction with cell surface proteins, followed by internalization through an endocytic mechanism (Loke, S. L., et al., Proc. Natl. Acad. Sci. USA 1989, 86, 3474-3478). These events lead to a punctate distribution of the oligonucleotides in intracellullar membrane-bound structures, which are thought to represent endosomes and lysosomes (Bennett, C. F., et al., Mol. Pharmacol. 1992, 41, 1023-1033). The sequestration of oligonucleotides into endosomal compartments may prevent their interaction with their target mRNA and hence decrease their activity.
One of the most commonly used enhancers is a mixture of a neutral lipid with a cationic lipid. The cationic lipid is thought to be the more crucial part of this mixture, as cationic lipids increase the activity of antisense oligonucleotides, whereas neutral lipids cannot. The mechanism by which cationic lipids increase the activity of antisense oligonucleotides is poorly understood. It was recently demonstrated that oligonucleotide-lipid complexes are taken into the cell via an endocytic mechanism and do not simply fuse with the plasma membrane (Zelphati, O. and Szoka, F. C., Jr., Pharmacol. Res. 1996, 13, 1367-1372). It is also known that cationic lipids enhance cellular accumulation of the oligonucleotide by increasing the amount that escapes from the endosomal pathway and thus has an opportunity to interact with its target mRNA (Bennett, C. F., et al., Mol. Pharmacol. 1992, 41, 1023-1033). While the mechanism by which an oligonucleotide is released from the endocytic compartments in order to gain access to its RNA target is not well characterized, models have been proposed (Zelphati, O. and Szoka, F. C., Jr., Proc. Natl. Acad. Sci. USA 1996, 93, 11493-11498). Fluorescence studies by Zelphati, O. and Szoka, F. C., Jr. have recently shown that when transfected with a cationic/neutral lipid mixture, oligonucleotides reached the nucleus while the neutral lipid stayed in punctate structures within the cytoplasm. These punctate structures were presumed to be endosome. However, the final destination of the cationic lipid was not examined in this study and whether the cationic lipid traffics together with the oligonucleotide to the nucleus was not determined.
Fully understanding the mechanism by which oligonucleotides are taken up by cells and released from endosomes will aid in the design of delivery vehicles to improve both in vitro and in vivo efficacy. Such improved designs are important in the investigation of the therapeutic utility of antisense oligonucleotides and to improve the dosing regimens for antisense therapeutics.
In Zelphati, O. and Szoka, F. C., Jr., cationic lipids were labeled with rhodamine and oligonucleotides were labeled with fluorescein. The mixture was introduced to cells. However, it is known that fluorescein has a quenching effect with respect to the cell culture medium, which results in loss of the fluorescent signal. Quenching also occurs using fluoroscein in acidic cellular environments, including endosomes, lysosomes and lipophilic cell surfaces. With quenching, it becomes more difficult to follow macromolecules, especially oligonucleotides, to such environments.
Thus, there remains a need for improved methods for tracking the distribution of carriers and macromolecules within a cell.